The long-term goal of this research is to elucidate the structure-function relationships in high density lipoproteins (HDL). In circulation, HDL solubilizes lipids, removes cholesterol from peripheral cells, activates the esterification of cholesterol, and delivers cholesterol esters to liver and steriodogenic tissues for metabolism and excretion. These functions of HDL, mediated by its major protein component, apolipoprotein A-I (apoA-I), underlie the antiatherogenic role of this lipoprotein class. In several key steps of HDL metabolism, apoA-I undergoes conformational changes to adapt to changing lipid contents of the HDL, to allow the binding of apoA-II to HDL, and possibly to mediate interaction with membranes and with lecithin cholesterol acyltransferase (LCAT). A putative "hinge" region has been implicated in the conformational changes of apoA-I. The specific aims of this project are to identify the helix(es) of apoA-I that are responsible for the "hinge" functions and the key amino acids that modulate the conformational adaptability of the "hinge" domain, and to study the structural and dynamic differences between the "closed" and "open hinge" forms of apoA-I. To identify the helix(es) involved in the "hinge" functions of apoA-I, we propose to construct and express in E. coli mutants of apoA-I with each of the candidate helixes replaced with the first or last helixes of apoA-I, which bind tightly to lipid and are not mobile. After structural studies of the mutants by spectroscopic (CD and fluorescence) methods and investigation of their lipid-binding properties, the apoA-I mutants in defined RHDL particles will be examined for their "hinge" functions: particle rearrangement, apoA-II binding, interaction with bilayer membranes, and LCAT activation. The identity of the "hinge" helix(es) will be confirmed and the structural rearrangements and their dynamics will be studied by constructing Cys mutants of apoA-I as sites for specific crosslinking or fluorescent labeling. The hydrophobicity, charge distribution, and role of Pro residues in the "hinge" helix(es) will be assessed by mutagenic substitution of individual amino acids. These studies are expected to localize the "hinge" region of apoA-I and to clarify its mechanism in several important functions of apoA-I.